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Plasma membranes were prepared from the P-glycoprotein expressing human breast cancer cell line MCF-7 ADR. [3H]Vinblastine bound to these membranes saturably with a Bmax of 24 pmol/mg of protein and KD of 23 nM. In contrast, membranes from the parent cells MCF-7 WT, which do not express P-glycoprotein, did not bind [3H]vinblastine with high affinity. Cytotoxics known to be transported by P-glycoprotein inhibited the binding of [3H]vinblastine, as did multidrug reversing agents including the 1,4-dihydropyridine, dexniguldipine-HCl (Ki, 15 nM). In dissociation kinetic experiments, dexniguldipine-HCl accelerated the dissociation of [3H]vinblastine from P-glycoprotein, indicating a negative heterotropic allosteric mechanism of action through a drug binding site distinct from that of vinblastine. Other 1,4-dihydropyridines tested also accelerated [3H]vinblastine dissociation from P-glycoprotein, however, multidrug reversing drugs of different chemical classes, including quinidine, verapamil and cyclosporin A did not. These results suggest that P-glycoprotein of MCF-7 ADR cell membranes possesses at least two drug acceptor sites which are allosterically coupled: receptor site-1 which binds vinca alkaloids, and receptor site-2 which binds 1,4-dihydropyridines such as dexniguldipine-HCl, which had the highest affinity of the tested derivatives.

Original publication

DOI

10.1016/0006-2952(94)00517-p

Type

Journal article

Journal

Biochem Pharmacol

Publication Date

16/06/1995

Volume

49

Pages

1851 - 1861

Keywords

ATP Binding Cassette Transporter, Subfamily B, Member 1, Allosteric Regulation, Cell Membrane, Dihydropyridines, Drug Resistance, Multiple, Drug Synergism, Humans, Ions, Kinetics, Nucleotides, Protein Binding, Tritium, Tumor Cells, Cultured, Vinblastine